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Recombinant human PrP91–231 folded into either α- or β-conformations8,9 was used to produce monoclonal antibodies in mice lacking PrP (Prnp0/0)10 that are intolerant to PrPC. ICSM 35, an immunoglobulin-γ2b (IgG2b) monoclonal antibody raised against β-PrP, with high affinity for both murine PrPc and PrPSc (Fig. 1a; A. Khalili-Shirazi, S.H. and J.C., unpublished data), recognizes a region between amino acid residues 91 and 110 (ref. 11). ICSM 18 (isotype IgG1), raised against α-PrP, recognizes residues 146–159 of murine PrP and has a lower affinity for PrPSc (Fig. 1a). FVB/N mice were challenged intraperitoneally (i.p.) with Rocky Mountain Laboratory (RML) scrapie brain homogenate derived from terminally scrapie-sick mice and treated with ICSM 35, ICSM 18 or isotype control antibodies BRIC 126 (IgG2b) and BRIC 222 (IgG1) by twice weekly i.p. injection (2 mg per injection) from 7 or 30 days post inoculation (p.i.). Enzyme-linked immunosorbent assay (ELISA) analysis after 30 days of antibody treatment revealed no significant differences between ICSM 35 or ICSM 18 antibody levels in the serum (Supplementary Information). Western blots of proteinase K-treated, phosphotungstic-acid-precipitated PrPSc from spleens of mice at 60 days p.i. revealed that treatment from 7 days p.i. with ICSM 35 or ICSM 18, but not with BRIC antibodies, markedly inhibited PrPSc accumulation in the spleen (Fig. 1b, c).

Figure 1: Anti-PrP antibodies inhibit splenic PrPSc protein levels.
figure 1

Western blots of proteinase K-digested, phosphotungstic-acid-precipitated PrPSc from spleens of mice 60 days p.i. with RML scrapie. Mice were inoculated i.p. except in h. Each lane contains PrPSc from an individual mouse. Mr, relative molecular mass; Ter, pooled splenic PrPSc from mice succumbing to terminal scrapie (195 ± 5 days p.i.). a, Immunoprecipitation of PrP from scrapie-infected mouse brain using ICSM or BRIC antibodies. PK, proteinase K. b, ICSM 18 and ICSM 35 induced substantial reductions in splenic PrPSc levels when treatment began from 7 days p.i., but this reduction was not seen in c, the BRIC-control-treated mice. d, ICSM 18 and ICSM 35 reduced splenic PrPSc when treatment began at 30 days p.i. e, Densitometry of PrPSc levels in the western blots. ICSM 18 induced greater reduction in PrPSc levels in spleens than ICSM 35, whereas BRIC 126 had no effect (asterisk, P < 0.001 compared with untreated spleens). f, ICSM 18 induced efficient clearance of PrPSc whether treatment began at 7 or 30 days p.i. ICSM 35 induced more efficient inhibition of PrPSc accumulation when treatment began at 7 days rather than 30 days p.i. g, ICSM 18 induced a dose-dependent reduction in PrPSc levels in spleens as determined by densitometry of western blots (asterisk, P < 0.001, ANOVA compared with control antibody (Ab) treatment). h, ICSM 18 and ICSM 35 inhibited splenic PrPSc accumulation in i.c. inoculated mice.

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Time course analysis of peripheral PrPSc accumulation in mice confirmed that PrPSc was detectable in the spleen at 7 days and plateaued by 30–40 days after peripheral challenge (data not shown) as previously reported12. Treatment of scrapie-infected mice with ICSM 35 or ICSM 18 from 30 to 60 days p.i. resulted in a substantial reduction in splenic PrPSc levels when compared with untreated controls (Fig. 1d) or mice treated with isotype control antibody (Fig. 1e). ICSM 18 reduced PrPSc levels by 99 ± 1% and 96 ± 3% (mean ± s.d., P < 0.001, analysis of variance (ANOVA)) after treatment from 7 or 30 days p.i., respectively (Fig. 1e). ICSM 35 also lowered PrPSc levels by 90 ± 8% and 75 ± 3% (P < 0.001, ANOVA) for the same treatment periods, whereas isotype control antibody did not alter PrPSc levels (Fig. 1e). Immunoblots probed with ICSM 35 instead of ICSM 18 as the primary antibody produced indistinguishable results (data not shown).

Treatment of mice from either 7 or 30 days p.i. with ICSM 18 consistently resulted in almost complete loss of detectable PrPSc in spleens by western blot (Fig. 1f). However, although ICSM 35 treatment from 7 days p.i. reduced splenic PrPSc levels with similar efficiency to ICSM 18, treatment with ICSM 35 from 30 days p.i. led to less complete inhibition of PrPSc replication (Fig. 1f). This may reflect differences in affinity or avidity of ICSM 35 and ICSM 18 for normal and disease-related PrP, as shown in Fig. 1a. Reduction in splenic PrPSc levels induced by ICSM 18 was dose-dependent (Fig. 1g). Analogous results were obtained when intracerebrally inoculated (i.c.) mice were treated with ICSM 18 or ICSM 35 (Fig. 1h). As ICSM 18 recognizes the PrP epitope at residues 146–159, these data are consistent with residues 132–156—which incorporate helix 1—of mouse PrP having a crucial role in prion replication6,7,13,14,15.

Tissue sections derived from formalin-fixed spleens at 60 days p.i. were examined by standard PrP immunohistochemistry using ICSM 18 and ICSM 35 (Fig. 2a–d). Both monoclonal antibodies were applied to adjacent splenic sections to ensure that antibody bound in vivo did not spuriously block the detecting antibody. ICSM 35 revealed intense labelling of PrPSc, primarily associated with germinal centres (Fig. 2a). PrPSc immunostaining was substantially reduced in spleens of ICSM 18- and ICSM 35-treated mice when labelled with ICSM 35 as primary antibody (Fig. 2b, c; see also Supplementary Information). ICSM 18 produced only weak staining of PrPSc in spleens from untreated, scrapie-infected mice and this staining was also diminished in both ICSM 35- and ICSM 18-treated mice (data not shown). As expected, PrPSc-positive germinal centres were not seen in splenic sections from mice that had not been challenged with RML prions (Fig. 2d). Bioassay of splenic homogenates from ICSM 35- and ICSM 18-treated mice showed a 1.5–3.5 and greater than 4 log reduction in infectious titres, respectively, compared with controls (Table 1).

Figure 2: Immunohistochemistry of spleens from antibody-treated mice.
figure 2

a–d, Immunohistochemical staining of spleens from ICSM 35-treated mice 60 days after i.p. inoculation with RML scrapie: a, immunostaining with ICSM 35 reveals strong PrP immunoreactivity (red) in follicle centres in spleens of scrapie-infected mice. b, After treatment with ICSM 18, very few follicles are immunoreactive for PrP. c, No positive follicles detectable after treatment with ICSM 35. d, No positive follicles are detectable in control spleens. Scale bar, 100 µm.

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Table 1 Effect of passive immunization with ICSM antibodies on spleen infectivity and survival of FVB/N mice inoculated with RML scrapie
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We then sought evidence for accompanying benefit to the health of the mice. Although treatment was ineffective if begun at the onset of clinical scrapie or after intracerebral challenge (Table 1), mice that were inoculated i.p. with RML scrapie and treated with ICSM 35 or ICSM 18 (2 mg twice weekly) from 7 and 30 days p.i. survived for much longer than untreated mice or mice treated with isotype control antibody (Table 1). The mean survival for untreated i.p.-inoculated mice was 197 ± 5 days. Mice treated with ICSM 18 or ICSM 35 from 7 or 30 days post i.p. challenge survived for more than 500 days p.i. (Table 1); an extension of the incubation period by at least 153% (P < 0.001, ANOVA). No clinical signs of scrapie (as described in the Methods) or weight loss (Supplementary Fig. 1) have yet been observed in these mice. Experiments are in progress to determine whether prion infection has been suppressed or eradicated in these animals and if the latter, what period of treatment is required to effect a cure.

Tissues were examined from mice lacking clinical signs of scrapie after treatment with ICSM 18 or ICSM 35 from 7 days p.i. In these mice (killed at 250 days p.i.), PrPSc was undetectable in the brain after either of the two treatments, and only low levels of PrPSc were seen in spleens of mice treated with ICSM 35 (Fig. 3a–d). Similarly, no PrPSc was observed in the brain after ICSM 35 treatment from 30 days p.i. and analysed at 230 days p.i. (data not shown). High levels of PrPSc were observed in brains and spleens from BRIC antibody-treated and untreated mice that succumbed to scrapie after i.p. inoculation (Fig. 3a–d). These findings were confirmed by histopathological analysis (data not shown), and bioassay of infectivity from these tissues is in progress.

Figure 3: PrPSc protein levels in mice with extended survival.
figure 3

Western blots of PrPSc in brain homogenates (a, c) or proteinase K (PK)-treated, phosphotungstic-acid-precipitated PrP from spleens (b, d). PrPSc was undetectable (a–c) or significantly reduced (d) in the brains and spleens of mice treated with ICSM 18 or ICSM 35 from 7 days p.i. and killed 250 days p.i. Mice treated with BRIC control antibody (Ab) succumbed to scrapie after 195 days p.i.

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We next examined whether passive immunization with anti-PrP monoclonal antibodies affected immune cell populations, as targeted depletion of PrP+ cell types could have contributed to loss of PrPSc detection in spleens of antibody-treated mice16,17. No differences were observed in splenic T- and B-cell populations between untreated scrapie-inoculated mice and antibody-treated mice (examined at 60 days p.i.) as determined by immunostaining of cryostat tissue sections or by flow cytometry (see Supplementary Fig. 2 and Supplementary Table 1). Follicular dendritic cells (FDCs) are an important site of PrPSc accumulation after peripheral prion infection17,18. Immunostaining of splenic sections from untreated mice revealed FDC-M1+ cells scattered throughout the germinal centres or in small clusters within the germinal centre (Supplementary Fig. 2). By contrast, ICSM or BRIC antibody-treated spleens often revealed larger numbers of FDC-M1+ cells clustered tightly within the germinal centre (Supplementary Fig. 2). These data clearly show that prolonged treatment with the anti-PrP antibodies does not induce depletion of the FDC cell type within the spleen, and strongly suggest that the substantial reduction in PrPSc levels observed in spleens treated with anti-PrP antibodies is probably due to direct inhibition of PrPSc production. This was further supported by unaltered PrPC protein levels in spleen homogenates from mice treated with ICSM 35 or ICSM 18 (Supplementary Fig. 3).

Our studies demonstrate that substantial peripheral prion replication can be effectively suppressed by passive immunization. Importantly, treatment began well after the onset of peripheral prion replication and in the case of the 30 days p.i. treatment group, during the plateau phase of PrPSc accumulation12. Continued treatment delayed onset of scrapie by more than 150% of the usual incubation period in wild type FVB/N mice. In contrast, almost all previous therapeutic interventions only show benefit if treatment is begun before, or very soon after the day of inoculation19,20,21,22,23,24, probably reflecting simple neutralization of the inoculum.

It is important to emphasize that passive transfer of these anti-PrP antibodies had no effect late in the incubation period when clinical signs had developed, probably reflecting inadequate translocation of anti-PrP antibody across the blood-brain barrier25. Furthermore, although we found no evidence for autoimmune reactions in mice, when treating humans infected with CJD or other prion diseases with humanized forms of these and other anti-PrP monoclonal antibodies, unwanted autoimmune side effects could develop. Despite these caveats, our findings are encouraging and provide an important impetus for pursuing prion immunotherapeutics.

Methods

Production of monoclonal antibodies

ICSM 35 and ICSM 18 monoclonal antibodies were produced as described11. ICSM and BRIC monoclonal antibodies were identically affinity purified from culture supernatant, concentrated, and stored as sterile solutions without vehicle protein at 4 °C. They were used undiluted or diluted in PBS before use in vivo.

Inoculation of FVB/N mice with RML prion inoculum

RML prions were passaged in FVB/N mice and prion inoculum was prepared from the brains of terminally sick mice (incubation time to terminal scrapie, 153 ± 4 days). Brains were homogenized in PBS (10% w/v) with 1% bovine serum albumin (BSA) using a Ribolyzer (Hybaid). The homogenate was spun for 5 min at 500g and supernatants pooled and frozen at -80 °C until use. The infectious titre of the pooled homogenate was determined as 8.1 log LD50 (lethal dose to 50% of animals) per g brain by infectivity assay with tga20 indicator mice26. FVB/N mice were inoculated i.c. with 30 µl or i.p. with 100 µl of 1% homogenate.

Infectivity bioassays

Assays were performed on 1% spleen homogenates. Aliquots of 30 µl were inoculated i.c. into groups of three or four tga20 mice per treatment regimen. Incubation time to terminal scrapie sickness was determined and infectivity titres were calculated by using the equation y = 11.45 - 0.088x (for RML 4.1), where y is the infectious titre (log LD50), and x is the incubation time (in days) to terminal disease26.

Passive immunization

Groups of mice were injected twice weekly through the intraperitoneal route with 2 mg (unless otherwise stated) ICSM 18, ICSM 35, IgG1 isotype control (BRIC 222 recognizing CD44; ref. 27) or IgG2b isotype control (BRIC 126 recognizing CD47; ref. 28) antibodies in PBS. Animals were monitored daily for clinical symptoms of scrapie29 and weighed weekly from 17 weeks after i.c. inoculation or 22 weeks after i.p. inoculation. Clinical signs in untreated mice were first observed approximately 4 weeks before terminal illness (day of death) and included coat ruffling/discoloration, progressive weight loss, bradykinesia (slow movement), tail rigidity, dystonia (clasp foot), kyphosis (hunched back), ataxia and stupor. Weights of scrapie-infected (untreated) mice decreased before terminal illness from 3 and 4 weeks in i.c.- and i.p.-inoculated mice, respectively (Supplementary Fig. 1). Confirmation of scrapie disease was performed by western blot analysis of PrPSc in brain tissue and in some cases by standard PrP immunohistochemistry.

Immunoprecipitation

Immunoprecipitation of PrP from murine brain tissues using ICSM and BRIC antibodies was performed as described11.

Western-blot analysis

Spleens were homogenized in PBS to 10% w/v and PrPSc was precipitated from 500 µl of homogenates using sodium phosphotungstic acid (NaPTA) as previously described30. PrPSc pellets were resuspended in 20 µl of 2% sarkosyl buffer, treated with proteinase K (50 µg ml-1, 50 min, 37 °C), boiled in sample buffer (5 min) and 15 µl aliquots (equivalent to about 2 mg spleen homogenate) were electrophoresed through 16% SDS–polyacrylamide electrophoresis gels. Brain homogenates were diluted to 1% (w/v) in PBS, treated with proteinase K (50 µg ml-1 for 60 min, 37 °C) and electrophoresed as for spleens. Proteins were transferred to polyvinylidene fluoride (PVDF) membrane by semi-dry blotting, blocked with TBST/5% non-fat milk, incubated with biotinylated ICSM 18 (0.1 µg ml-1) and developed by enhanced chemiluminescence (Amersham). Semi-quantification was performed by densitometric analysis using MacBas version 2.5 software. At least three to four mice were examined from each treatment group. Bars on graphs are standard deviations. To standardize the PrPSc signal between blots, 10 µl of PrPSc precipitated from pooled spleens of terminal (Ter) scrapie-affected mice was loaded on each gel. Densitometric measurement of PrPSc from treated and untreated spleens was compared to the standard PrPSc sample and adjusted to relative intensities.

Histology and immunohistochemistry

For PrPSc immunohistochemistry, spleens and brains were fixed in 10% formalin. Prion infectivity was inactivated by immersion in 98% formic acid and postfixed in formalin for 24 h. Tissues were dehydrated in graded alcohols and xylene, embedded in paraffin, sections cut at 3 µm nominal thickness and stained with haematoxylin and eosin. After antigen retrieval by microwave treatment for 15 min, sections were immunostained with biotinylated ICSM 18 or ICSM 35 on a Ventana automated staining apparatus.